Intensified constructed wetlands for the treatment of municipal wastewater: experimental investigation and kinetic modelling.

This study reports organics and nutrient removal performances of the intensified constructed wetlands, i.e., tidal flow-based microbial fuel cell (MFC) and tidal flow wetlands that received municipal wastewater. The wetland systems were filled with organic (coco peat, biochar) or waste (Jhama brick, steel slag) materials, planted with Phragmites australis or Chrysopogon zizanioides (Vetiver) species, and operated under three flood periods: 8, 16, 24 h. Input ammonia nitrogen (NH3-N), total nitrogen (TN), phosphorus (P), chemical oxygen demand (COD), and biochemical oxygen demand (BOD) load across the wetland systems ranged between 3-27, 12-78, 0.1-23, 36-1130, and 11-281 g/m2day, respectively; mean removal percentages were 60-83, 74-84, 95-100, 94-98, and 93-97%, respectively, throughout the experimental run. The wetland systems achieved similar organics and P removals; operational and media variation did not influence removal kinetics. All wetland systems achieved the highest TN removal (76-87%) when subjected to 24-h flood period. TN removal performances of waste material-based wetlands were comparable to organic media-based systems. Tidal flow-based MFC wetlands achieved better TN removal than tidal flow wetlands because of supplementary electron production through fuel cell-based organics degradation kinetics. Maximum power production rates across the tidal flow-based MFC wetlands ranged between 53 and 57 mW/m2. Monod kinetics-based continuous stirred tank reactor (CSTR) models predicted NH3-N, TN, and COD removals (in wetland systems) more accurately. Kinetic models confirmed the influence of substrate (i.e., pollutant) and environmental parameters on pollutant removal routes.

Pollutant removal from landfill leachate employing two-stage constructed wetland mesocosms: co-treatment with municipal sewage.

Constructed wetlands are low-cost, natural technologies that are often employed for the treatment of different types of wastewater. In this study, landfill leachate and municipal wastewater were co-treated by the three parallel two-stage Phragmites- or Vetiver-based constructed wetland mesocosms. Two-stage wetland mesocosms included vertical flow (VF) units as the first stage, followed by horizontal flow (HF)/surface flow (SF)/floating treatment (FT) units. VF and HF wetland mesocosms were filled with gravel, steel slag, concrete block, and intermittent carbon-saturated ceramic filters as substrates. Mean input nitrogen, organics, and phosphorus load across first stages were 75 g N/m2 day, 283 g COD/m2 day, 88 g BOD/m2 day, and 10 g P/m2 day, respectively. N and P accumulation rate was not substantial (< 10%) with respect to total removal in most wetland mesocosms. Gravel-based VF wetland mesocosm achieved better NH4-N and BOD removal (55-59%) during landfill leachate treatment phase, when compared with co-treatment periods (12-52%). Slag-concrete- and ceramic filter-based VF wetland mesocosms maintained stable NH4-N and BOD removals; the former wetland mesocosm was the most efficient VF unit (than other two wetland mesocosms) due to media characteristics. Media-based adsorption accelerated P removal (93%) in slag-concrete-based VF wetland mesocosm. Carbon scarcity limited denitrification in all VF wetland mesocosms; removal of TN was < 32%. Second stage wetland mesocosms achieved higher nitrogen (85-92%), organics (66-90%), and phosphorus (97-100%) removals regardless of operational variations; low input load, long retention time, media, and rhizosphere enhanced removal performances, particularly in HF and FT wetland mesocosms. In general, this study demonstrates potential application of two-stage wetland mesocosms for landfill leachate treatment or co-treatment with municipal sewage.

Two-stage constructed wetland systems for polluted surface water treatment.

Two pilot scale wetland systems were studied for the removal of organics, nitrogen, phosphorus and coliform from polluted surface water. Each system consisted of two units: a vertical flow (VF) wetland packed with construction materials gravel, brick or organic sugarcane bagasse, followed by a surface flow (SF) or floating treatment (FT) wetland. All wetland units were planted with Phragmites. The wetland systems were operated under constant and shock hydraulic load (HL) periods. Input COD, N, P loadings ranged between 61 and 2181, 7-1040, 2-194 g/m2d, respectively across first stages of each system. Mean removal percentages ranged between 39 and 97, 11-83, 20-100% and 4-85, 16-86, 1.4-100% across first and second stage wetlands, respectively. Mass balance analyses revealed ≤7% N and ≤14% P accumulation in plants; as such, microbial and adsorption kinetics controlled removal dynamics. Nitrification was the limiting nitrogen removal factor in first stage wetlands; organic carbon was supplied by the employed media. Aerobic organics removal and nitrification were diminished during initial stage of shock load periods. In contrast, second stage SF and FT wetlands showed stable removal performances under similar conditions. Resuspension of settled particles decreased removal performance in second stage wetlands, as shock periods progressed toward final stage. Coliform mortality was increased in second stage wetlands. Physico-chemical properties of brick materials in construction material based VF wetland and hanging root volume inside the water column of FT wetland supplemented removal performance. In general, this study provides evidence on potential application of constructed wetlands for polluted surface water treatment.

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients.

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.

Identification of guanine nucleotide-binding protein γ-7 as an epigenetically silenced gene in head and neck cancer by gene expression profiling.

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. Aberrant hypermethylation in the promoter region of some known or putative tumor suppressor genes occurs frequently during the development of various types of cancer including head and neck squamous cell carcinoma (HNSCC). In this study we used an expanded mRNA expression profiling approach followed by microarray expression analysis to identify epigenetically inactivated genes in HNSCC. Two HNSCC cell lines were treated with 5-aza-2'-deoxycytidine followed by microarray analysis to identify epigenetically silenced genes in HNSCC. We found 1,960, 614 and 427 genes were upregulated in the HNSCC cell lines JHU-012, JHU-011 and the combination of both cell lines, respectively. HNSCC tumor and normal mucosal samples were used for gene profiling by a 47K mRNA gene expression array and we found 7,140 genes were downregulated in HNSCC tumors compared to normal mucosa, as determined by microarray analysis, and were integrated with cell line data. Integrative analysis defined 126 candidate genes, of which only seven genes showed differential methylation in tumors and no methylation in normal mucosa after bisulfite sequencing. Following validation by QMSP, one gene, guanine nucleotide-binding protein γ-7 (GNG7), was confirmed to be highly methylated in tumors and unmethylated in normal mucosal and salivary rinse samples demonstrating cancer-specific methylation in HNSCC tissues. TXNIP and TUSC2 were partially methylated in tumors and normal salivary rinses but unmethylated in normal mucosa. We concluded that GNG7 is a highly specific promoter methylated gene associated with HNSCC. In addition, TXNIP and TUSC2 are also potential biomarkers for HNSCC.

Comparison of promoter hypermethylation pattern in salivary rinses collected with and without an exfoliating brush from patients with HNSCC.

BACKGROUND: Salivary rinses have been recently proposed as a valuable resource for the development of epigenetic biomarkers for detection and monitoring of head and neck squamous cell carcinoma (HNSCC). Both salivary rinses collected with and without an exfoliating brush from patients with HNSCC are used in detection of promoter hypermethylation, yet their correlation of promoter hypermethylation has not been evaluated. This study was to evaluate the concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush from patients with HNSCC. METHODOLGY: 57 paired salivary rinses collected with or without an exfoliating brush from identical HNSCC patients were evaluated for promoter hypermethylation status using Quantitative Methylation-Specific PCR. Target tumor suppressor gene promoter regions were selected based on our previous studies describing a panel for HNSCC screening and surveillance, including P16, CCNA1, DCC, TIMP3, MGMT, DAPK and MINT31. PRINCIPAL FINDINGS: In salivary rinses collected with and without brush, frequent methylation was detected in P16 (8.8% vs. 5.2%), CCNA1 (26.3% vs. 22.8%), DCC (33.3% vs. 29.8%), TIMP3 (31.6% vs. 36.8%), MGMT (29.8% vs. 38.6%), DAPK (14.0% vs. 19.2%), and MINT31 (10.5% vs. 8.8%). Spearman's rank correlation coefficient showed a positive correlation between salivary rinses collected with and without brush for P16 (ρ = 0.79), CCNA1 (ρ = 0.61), DCC (ρ = 0.58), TIMP3 (ρ = 0.10), MGMT (ρ = 0.70), DAPK (ρ = 0.51) and MINT31 (ρ = 0.72) (P<0.01). The percent agreement of promoter methylation between salivary rinses with brush and without brush were 96.5% for P16, 82.5% for CCNA1, 78.9% for DCC, 59.7% for TIMP3, 84.2% for MGMT, 84.2% for DAPK, and 94.7% for MINT31. CONCLUSIONS: Our study demonstrated strong correlations of gene promoter hypermethylation between salivary rinses collected with and without an exfoliating brush. Salivary rinse collection without using an exfoliating brush may offer a cost effective, rapid, non-invasive, and reliable means for development of epigenetic salivary rinse biomarkers.

Detection of TIMP3 promoter hypermethylation in salivary rinse as an independent predictor of local recurrence-free survival in head and neck cancer.

PURPOSE: To validate a panel of methylation-based salivary rinse biomarkers (P16, CCNA1, DCC, TIMP3, MGMT, DAPK, and MINT31) previously shown to be independently associated with poor overall survival and local recurrence in a larger, separate cohort of patients with head and neck squamous cell carcinoma (HNSCC). EXPERIMENTAL DESIGN: One hundred ninety-seven patients were included. All pretreatment saliva DNA samples were evaluated for the methylation status of the gene promoters by quantitative methylation-specific PCR. The main outcome measures were overall survival, local recurrence-free survival, and disease-free survival. RESULTS: In univariate analyses, the detection of hypermethylation of CCNA1, MGMT, and MINT31 was significantly associated with poor overall survival; the detection of hypermethylation of TIMP3 was significantly associated with local recurrence-free survival; and the detection of hypermethylation of MINT31 was significantly associated with poor disease-free survival. In multivariate analyses, detection of hypermethylation at any single marker was not predictive of overall survival in patients with HNSCC; detection of hypermethylation of TIMP3 in salivary rinse had an independent, significant association with local recurrence-free survival (HR = 2.51; 95% CI: 1.10-5.68); and none of the studied markers was significantly associated with disease-free survival. CONCLUSION: The detection of promoter hypermethylation of the seven genes in salivary rinse as an independent prognostic indicator of overall survival in patients with HNSCC was not validated. Detection of promoter hypermethylation of TIMP3 in pretreatment salivary rinse is independently associated with local recurrence-free survival in patients with HNSCC and may be a valuable salivary rinse biomarker for HNSCC recurrence.

Detection of promoter hypermethylation in salivary rinses as a biomarker for head and neck squamous cell carcinoma surveillance.

PURPOSE: Hypermethylation of tumor suppressor gene promoters has been found in head and neck squamous carcinoma (HNSCC) and other solid tumors. We evaluated these alterations in pretreatment salivary rinses from HNSCC patients by using real-time quantitative methylation-specific PCR (Q-MSP). EXPERIMENTAL DESIGN: Pretreatment saliva DNA samples from HNSCC patients were evaluated for patterns of hypermethylation by using Q-MSP. Target tumor suppressor gene promoter regions were selected based on a previous study describing a screening panel for HNSCC in a high-risk population subjects. The selected genes were: DAPK, DCC, MINT-31, TIMP-3, p16, MGMT, CCNA1. RESULTS: We analyzed the panel in a cohort of 61 HNSCC patients. Thirty-three of the analyzed patients (54.1%) showed methylation of at least one of the selected genes in the saliva DNA. Pretreatment methylated saliva DNA was not significantly associated with tumor site (P = 0.209) nor clinical stage (P = 0.299). However, local disease control and overall survival were significantly lower in patients presenting hypermethylation in saliva rinses (P = 0.010 and P = 0.015, respectively). Multivariate analysis confirmed that this hypermethylation pattern remained as an independent prognostic factor for local recurrence (HR = 12.2; 95% CI = 1.8-80.6; P = 0.010) and overall survival (HR = 2.8; 95% CI = 1.2-6.5; P = 0.016). CONCLUSIONS: We were able to confirm an elevated rate of promoter hypermethylation in HNSCC saliva of patients by using a panel of gene promoters previously described as methylated specifically in HNSCC. Detection of hypermethylation in pretreatment saliva DNA seems to be predictive of local recurrence and overall survival. This finding has potential to influence treatment and surveillance of HNSCC patients.

KIF1A and EDNRB are differentially methylated in primary HNSCC and salivary rinses.

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. In this study, we aimed to evaluate to the utility of aberrant promoter hypermethylation for detection in a panel of 10 genes (KIF1A, EDNRB, CDH4, TERT, CD44, NISCH, PAK3, VGF, MAL and FKBP4) in head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. We investigated methylation of the gene promoters by bisulfite modification and quantitative methylation-specific PCR (Q-MSP) in a preliminary study of a limited cohort of salivary rinses from healthy subjects (n = 61) and patients with HNSCC (n = 33). The methylation status of 2 selected genes (EDNRB and KIF1A) were then analyzed in 15 normal mucosa samples from a healthy population, 101 HNSCC tumors and the corresponding salivary rinses from 71 out of the 101 HNSCC patients were collected before treatment. The promoter regions of CDH4, TERT, VGF, MAL, FKBP4, NISCH and PAK3 were methylated in normal salivary rinses while no methylation of CD44 was observed in either normal salivary rinses or tumor samples. However, KIF1A and EDNRB were methylated in 98 and 97% of primary HNSCC tissues respectively and were only methylated in 2 and 6.6% of normal salivary rinses. In addition, KIF1A and EDNRB were methylated in 38 and 67.6% of salivary rinses from HNSCC patients, respectively. Promoter hypermethylation of KIF1A and EDNRB is a frequent event in primary HNSCC, and these genes are preferentially methylated in salivary rinses from HNSCC patients. KIF1A and EDNRB are potential biomarkers for HNSCC detection.